A hallmark of human glioblastomas is that oncogenic signalings stimulated by overexpressed genes such as EGFR and EGFRvIII are aberrantly active, rendering these tumors highly malignant, and inherently resistant to combination therapies, resulting in an extremely poor prognosis of patients with malignant gliomas. A major barrier of this clinical challenge is the lack of a complete understanding of the mechanisms underlying the aggressiveness of these tumors and ineffectiveness of therapies for gliomas. The goal of this project is to define the role of ESDN, endothelial and smooth muscle cell-derived neuropilin-like protein (other names, DCBLD2 and CLCP1) in tumorigenesis and therapy-resistance of malignant human glioblastomas. ESDN is expressed at high levels in vascular injury, metastatic lung cancer and breast cancer. Our preliminary studies show that the ESDN gene is amplified in ~50% of clinical glioma specimens and preferentially up-regulated in classical and mesenchymal subtypes of glioblastomas that overexpress EGFR and EGFRvIII. Knockdown of ESDN in tumor xenografts significantly suppressed EGFRvIII-promoted glioma growth and invasion in the brain of animals. Mechanistically, EGFRvIII and EGF induce tyrosine phosphorylation (p-Y) of Y621 and Y750 of ESDN that activate TRAF6-Akt- and CrkII-Rac1- signaling and promote glioblastoma cell survival and invasion. Based on these observations, in this project, we propose a research plan directed at elucidating mechanisms by which ESDN augments EGFRvIII-stimulated glioma tumorigenesis and exploring therapeutic application of targeting ESDN to inhibit EGFRvIII-expressing brain gliomas. We will first investigate how ESDN affects EGFRvIII-stimulated glioma growth and invasion through dissociation of TRAF6 and binding of CrkII to specific tyrosine residues of ESDN that are phosphorylated by EGF and EGFRvIII stimulation. We will then determine clinical significance of the induced p-Y of ESDN with activation of oncogenic EGFR/EGFRvIII signaling pathways in primary glioma tumor specimens (Aims 1 and 2). Using short-term cultured primary glioblastoma (GBM) xenograft and an induced de novel brain glioma models, we will explore pre-clinical approaches to assess the impact of inhibition of ESDN on the efficacies of chemotherapeutic agents, cisplatin and temozolomide on EGFR- and EGFRvIII-expressing glioblastomas in the brain of animals (Aim 3). Therefore, this proposal reveals an unrecognized signaling path by which EGFR and EGFRvIII stimulate TRAF6-Akt- and CrkII-signaling through ESDN, thereby promoting glioma growth and invasion. Elucidation of this novel mechanism holds promise to a better understanding of the oncogenic signaling-promoted glioma tumorigenesis and to overcome the insidious glioma resistance to current therapies. This project also addresses an urgent challenge in neuro-oncology and offers an enormous potential for developing novel therapies that could prove effective, thus eliminating the major barrier in treating patients with malignant glioblastomas.